This application is a 371 of PCT/EP99/03771 filed May 31, 1999.
The present invention relates to the use of selenium and/or a derivative thereof in combination with a cytostatic or a mixture of cytostatics.
The chemical element selenium is a trace element which is essential for humans and animals and influences above all oxidative processes as well as thyroxine metabolism. In humans it could be detected that the enzyme glutathione peroxidase and the selenoprotein P found in plasma contain selenium in the form of the amino acid selenocysteine. The selenium-containing glutathione peroxidase forms part of the antioxidative protective system of the mammalian cell. In the presence of sufficient amounts of substrate, i.e. reduced glutathione, glutathione peroxidase converts a multitude of different hydroperoxides into corresponding alcohols. It could be demonstrated that the integrity of cellular and subcellular membranes decisively depends on the intactness of the glutathione peroxidase system. Selenium as part of glutathione peroxidase can reduce the lipid peroxidation rate and the resulting membrane damage.
In animals the type-I iodothyronine-5xe2x80x2-deiodase was recently characterized as a selenium-containing enzyme. In the thyroid, liver and lung of humans, iodothyronine deiodase also converts thyroxine (T4) into triiodothyronine (T3), the active thyroid hormone. In the case of selenium deficiencies, e.g. phenylketonuria and cystic fibrosis, increased T4 values could be detected at a simultaneously reduced T3 level. By the administration of sodium selenite (Na2SeO3) the thyroid metabolism is normalized again.
As a further selenium-dependent enzyme, a human thioredoxin reductase from lung cells was recently described to contain selenium as a cofactor (Tamura and Stadtman, 1996, Biochemistry, Proc. Natl. Acad. Sci., 93: 1006-101 1). The enzyme could so far be isolated from T cells, lung tissue and placenta (Gladyshev et al., 1996, Biochemistry, Proc. Natl. Acad. Sci., 93: 6146-6151). The selenium-dependent enzyme thioredoxin reductase reduces thioredoxin. Thioredoxin is overexpressed in a number of tumors, and some experimental studies have shown that thioredoxin contributes to the growth and malign transformation of some human cancer cells. The enzyme thioredoxin reductase therefore plays a role in the regulation of the growth of normal and cancer cells.
Proof of the pathophysiological relevance of the selenium-dependent reactions has been furnished by observation of selenium deficiency symptoms in humans and in animals. Deficiency of this trace element intensifies oxidatively or chemically induced liver damage and the toxicity of heavy metals such as mercury and cadmium.
In humans the Keshan disease, an endemically occurring cariomyopathy, and the so-called Kaschin-Beck disease, also an endemically occurring osteoathropathy with strong deformations of the joints, are described as selenium deficiency symptoms. Clinically manifested selenium deficiency was also observed as a consequence of long-term parenteral feeding and of balanced diets. Cardiomyopathies and myopathies of the skeletal muscles as well as a shift in the T3/T4 ratio were above all observed.
Epidemiological studies hint at an inverse correlation between blood-selenium level and the incidence of cardiovascular diseases (cardiomyopathies, arteriosclerosis, myocardial infraction) and tumor diseases, in particular of the digestive system, breast and liver. Reduced selenium levels in plasma may be present in patients with renal insufficiency and in the case of gastrointestinal diseases. Selenium deficiency can be detected through a reduced selenium level in whole blood or plasma and a reduced glutathione peroxidase activity in whole blood, plasma or thrombocytes.
Selenium substitution in the case of deficiency symptoms activates reactions of the immune defense, in particular unspecific, cell-bound and humoral reactions. The selenium-containing glutathione peroxidase influences leukotriene, thromboxane and prostacyclin metabolism. The immunomodulatory effects of selenium-containing compounds are listed in the following:
Stimulation of lymphocyte proliferation
Activation of cytotoxic T cells and NK cells
Increase in interleukin-2 receptor expression
Selective reduction of the number of T suppressor cells
Increase in interferon-xcex3 synthesis
general decrease in infection frequency
Selenium in the form of selenite (SeO32xe2x88x92) is not directly incorporated into proteins. In blood, selenite is first mainly taken up by erythrocytes and enzymatically reduced to selenium hydrogen. Selenium hydrogen serves as a central selenium pool for excretion and for the targeted incorporation into selenoproteins. In this reduced form selenium is bound to plasma proteins which migrate into the liver and other organs. The plasmatic secondary transportation starting from the liver into the glutathione peroxidase-synthetized target tissue probably takes place in the form of a selenocysteine-containing P selenoprotein. The further metabolic course of selenoprotein biosynthesis has so far only been known from prokaryotic model organisms. In these organisms selenocysteine is specifically incorporated-into the peptide chain of the glutathione peroxidase in the course of the translation.
Excessive selenium hydrogen in humans is metabolized through methylselenol and dimethylselenide to trimethylselenonium ion, the main excretion product. After oral application selenite is predominantly absorbed from the small intestine. The intestinal absorption of sodium selenite is not regulated homeostatically. Depending on the concentration and on additives, it is between 44% and 89%, sometimes over 90%. The amino acid cysteine promotes the sodium selenite absorption.
Organic selenium compounds must first be converted into selenium hydrogen before they are available for the synthesis of selenoproteins. Instead of methionine, selenomethionine, which is mainly contained in food, can also be unspecifically incorporated statistically in the case of protein biosynthesis into proteins that do not contain selenium.
The total amount of selenium in the human body is between 4 mg and 20 mg in a well-balanced selenium metabolism. Selenium is excreted in humans via urine, faeces and lung, depending on the dose applied. Selenium is primarily excreted renally in the form of the above-mentioned trimethylselenonium ions.
In humans acute selenium intoxications have hardly been described up to now. Garlic-like breadth, tiredness, queasiness, diarrhea and abdominal pain are regarded as signs of an acute overdosage. In humans, a safe maximum daily intake of selenium of 820 xcexcg was inferred from observations regarding the chronic toxicity of selenium, while a dosage of up to 500 xcexcg per day is also considered to be harmless in sensitive persons. As clinical signs of endemically occurring selenosis, alopecia, brittleness of the finger nails, skin alterations and disorders in the nerve system were observed in a study carried out in China after a daily supply of 3200-6700 xcexcg selenium. In various species a decreased reproductive capacity because of a reduced motility of spermatozoons was described as a symptom of selenosis.
In a dose/escalation study, between 10 and 50 mg selenium were infused in the form of sodium-selenite pentahydrate in tumor patients. Within 30 minutes the selenium level in plasma rose from 200 xcexcg/l to 1200 xcexcg/l after administration of 10 mg selenium as sodium selenite. After 8 and 16 hours the plasma selenium decreased to 770 xcexcg/I and 430 xcexcg/I, respectively. After 24 hours the selenium level in plasma had again reached its initial value. Gastrointestinal toxicity was observed starting from about 20 mg selenium as sodium selenite and was reversible after the administration of the preparation had been stopped (Rxc3x6hrer H., 1989, Erfahrungsheilkunde 38: 10a, 761).
As counter-measures in the case of intoxication, gastric lavage, forced diuresis, or highly dosed vitamin C administrations are possible. In the case of an extreme overdosage (1000 to 10000 times), the attempt can be made to eliminate selenite by dialysis.
In humans, the trace element selenium is predominantly taken in by consumption of yolk, fish and meat, in particular chicken and pork, as well as innards. The minimum selenium supply required for humans depends on the chemical form of the consumed element and on the composition of the diet in which it is present. In China, experiments revealed an amount of 15-20 xcexcg selenium a day to be sufficient as protection against endemic selenium deficiency diseases. The National Research Council (NRC) of the USA recommends a daily supply of 70 xcexcg selenium for males and 55 xcexcg selenium for females. In former times (up to 1989) the NCR regarded daily amounts of 50-200 xcexcg selenium as adequate and harmless. The German Society for Alimentation recommends 20-100 xcexcg selenium per day.
The daily average selenium supply, 2/3 covered by the supply of animal protein, is 38 xcexcg for women and 47 xcexcg for men in the old federal states of Germany. By contrast, in the territory of the new federal states of Germany, values of 20-25 xcexcg selenium were determined. These figures demonstrate that the nutritive selenium supply in Germany is not always covered. The risk of an insufficient supply with selenium exists especially in situations of increased demands (e.g. pregnancy and lactation period), in persons exposed to heavy metals and oxidants, in patients with gastrointestinal complications (e.g. chronically inflammatory bowel diseases) and in parenterally fed persons or persons observing special diets (e.g. in the case of phenylketonuria).
Epidemiological studies have shown that a low selenium intake and correspondingly low selenium levels in plasma are connected with an increased incidence of a variety of cancers in humans (Glattre et al., 1989, Int. J. Epedemiol., 18:45-49; Knekt et al., 1990; J. Natl. Cancer Inst., 82:864-868; Burney et al., 1997, J. Clin. Nutr., 49:895-900). Selenium has also been shown to markedly inhibit the growth of different tumor cells in vitro in high dose levels (20-200 xcexcM), including human mammary, ovarian and colon tumor cells. (Yan et al., 1991; Biol. Trace Elem. Res., 30:145-162; Chen et al., 1995, FASEB J., 9(3): A159; Nano et al., 1989, Biol. Trace Elem. Res. 20: 31-43; Stewart et al., 1997, Cancer Lett., 117:35-40). By contrast, several scientists reported on the growth stimulating effect of small amounts of sodium selenite (0.001-1 xcexcM) on various tumor cells incubated under serum-free culture conditions (Nano et al., 1989, Biol. Trace Elem. Res., 20:31-43; Goiczewski and Frenkel, 1989, Biol. Trace Elem. Res. 20:115-126). It has also been observed that organic selenium compounds have a preventive effect on the tumor development of mammary carcinomas in mice and rats (El-Bayoumy et al., 1995, J. Cell. Biochemistry, Annex 22: 29-100). The mechanism by which selenium influences tumor proliferation or regression is mainly unknown. However, it seems that induction of DNA strand breaks and apoptosis due to selenium and/or selenium metabolites like selenodiglutathione and hydrogen selenite as well as the formation of selenoproteins such as glutathione peroxidase and thioredoxin reductase play an important role (Thompson et al., 1994, Carcinogenesis 15:183-186; Wu et al., 1995, Carcinogenesis 16: 1579-1584; Lu et al., 1994, Biochem. Pharmacol., 47:1531-1535; Milner, 1985, Fed. Proc., 44: 2568-2572; Schrauzer, 1992, Biol. Trace Elem. Res., 33:51-62; Gallegos et al., 1997, Cancer Res., 75:4965-4979). For example, enhancing thioredoxin reductase activity by selenium could reduce cellular thioredoxin concentration and therefore play a role in the growth regulation of cancer cells (Gallegos et al., 1997, Cancer Res., 75: 4965-4979).
In combination experiments it has been observed that the administration of small amounts of selenium or selenium-containing compounds together with cytostatics does not decrease the antitumor effect, but can considerably reduce the side effects caused by cytostatics, for instance nephrotoxicity or cardiotoxicity.
While quite efficient therapeutic methods could already be developed for some types of cancer (the mortality rate following colon cancer disease could e.g. be reduced by about 17% between 1992 to 1993), there has so far been no or only a very inadequate therapy for the great majority of types of cancer.
Apart from the operative removal of the tumor and radiation therapy, chemotherapy is considered the so far most efficient therapeutic method. Chemotherapeutic drugs can substantially be divided into the following four groups: antimetabolites, topoisomerase inhibitors, alkylating agents and plant alkaloids, the three first-mentioned groups preventing a correct replication of the genetic substance, and the last-mentioned group having a mitosis-inhibiting effect. In the treatment of above all solid tumors the effect of cytostatics is most of the time not sufficient for curatively treating tumors.
It is therefore the object of the present invention to provide a possibility of enhancing the effect of antitumor drugs and to provide said drugs in a suitable form of administration.
This object is achieved by using selenium and/or at least one selenium compound for enhancing the effect of a cytostatic or a mixture of cytostatics.
Furthermore, this object is achieved by providing a kit comprising selenium and/or at least one selenium compound and a cytostatic or a mixture of cytostatics as a combination preparation for simultaneous, separate or sequential application in cytostatic therapy.
The present invention relates to the use of selenium and/or at least one selenium compound for enhancing the effect of a cytostatic or a mixture of cytostatics. The following examples will demonstrate that in vitro a simultaneous treatment with the above-mentioned components surprisingly yields a distinct synergistic, i.e. superadditive, antitumor effect.
Organic and inorganic selenium compounds are used for combination with cytostatics. In a preferred embodiment use is made of an organic selenium compound. The use of organic selenium compounds is to reduce toxicity in comparison with inorganic selenium compounds with simultaneous or improved antitumor efficiency. Particularly preferred are the selenium amino acids selenomethionine and selenocysteine as well as the compound phenylenebis(methylene)selenocyanate as well as derivatives thereof (El-Bayonmi et al., 1995, J. Cell. Biochemistry, Annex 22: 92-100). The last-mentioned compound inhibits thymidine kinase in human mammary carcinoma cell lines. Furthermore, it has been reported that said compound can trigger the inhibition of cell growth and the induction of cell death by apoptosis.
Furthermore, a selenium oxide is preferred as the selenium compound for enhancing the effect of a cytostatic or a mixture of cytostatics. In a particularly preferred embodiment, the selenium compound is a salt of SeO2, e.g. the salt Na2SeO3.
The cytostatic that is used together with selenium or a selenium compound may be a mitosis-inhibiting cytostatic. Examples of said group are inter alia substances, such as vinblastine and vinorelbine.
The cytostatic used together with selenium or a selenium compound may also be a cytostatic inhibiting nucleic acid synthesis, for example methotrexate and fluorouracil, which belong to the group of antimetabolites, or the topisomerase inhibitor topotecan, mRNA synthesis inhibitors such as doxorubicin, or alkylating agents such as cyclophosphamide and chlorambucil. The following table gives examples of different cytostatics, in the order of their modes of action, which are suited for administration together with selenium compounds. Combinations of several different cytostatics can also be used together with selenium compounds.
A preferred cytostatic inhibiting nucleic acid synthesis is gemcitabine. A further, also preferred, cytostatic that inhibits nucleic acid synthesis is the compound mitomycin C. The structural formulae of said two compounds are shown in the following: 
Mitomycin C belongs to the group of alkylating agents. Upon reduction of the quinone unit, methanol is released, which facilitates opening of the aziridine ring to form an alkylating metabolite. A further alkylating molecule is formed by chemical or enzymatic separation of the carbamate side chain. Moreover, the reduction of the quinone unit is connected with the formation of reactive oxygen molecules, which also have alkylating potency. The antitumor effect of mitomycin is mainly due to the alkylation of DNA.
Gemcitabine is a pyrimidine antimetabolite. After cellular uptake, it is metabolized to 2xe2x80x2,2xe2x80x2-difluoro-deoxycytidinetnphosphate. Incorporation of gemcitabine into DNA terminates DNA strand synthesis, so that cell division is no longer possible.
Gemcitabine and mitomycin C have different modes of action, but both compounds interact directly with cellular DNA, leading to errors or discontinuance of DNA replication.
To enhance the effect of a cytostatic or the combination of several cytostatics, selenium and/or at least one selenium compound is used in a concentration of 0.1 mg/kg body weight to 1.25 mg/kg body weight, and the cytostatic is used in a concentration of 2 mg/M2 body surface to 240 g/m2 body surface. The preferred concentration of selenium or a selenium compound is in a range of 0.1 mg/kg body weight to 0.3 mg/kg body weight, and that of the cytostatic is in a range of 20 mg/M2 body surface to 1000 mg/m2 body surface.
The use of said combination in cytostatic therapy is particularly preferred.
Furthermore, the invention provides a kit which comprises selenium and/or at least one selenium compound and a cytostatic or a mixture of cytostatics as combination preparation for simultaneous, separate or sequential application in cytostatic therapy. It is preferred that the selenium compound contained in the kit is an organic selenium compound. Particularly preferred organic selenium compounds are the selenium amino acids selenomethionine and selenocysteine as well as the compound phenylene-bis(methylene)selenium cyanate. Moreover, a selenium oxide is preferred in a further embodiment. Particularly preferred is a salt of SeO2, e.g. Na2SeO3.
Furthermore, the above-mentioned kit may contain a cytostatic which is a mitosis-inhibiting cytostatic, e.g. selected from the above-mentioned compounds. Furthermore, the cytostatic may also be a cytostatic inhibiting nucleic acid synthesis. The compounds gemcitabine and mitomycin C are here particularly preferred cytostatics inhibiting nucleic acid synthesis.
The kit according to the invention contains selenium and/or at least one selenium compound in a concentration of 0.1 mg/kg body weight to 1.25 mg/kg body weight and a cytostatic as described above in a concentration of 2 mg/M2 body surface to 240 mg/M2 body surface. A concentration range of 0.1 mg/kg body weight to 0.3 mg/kg body weight is here preferred for selenium or a selenium compound, and a concentration range of 20 mg/kg body weight to 1000 mg/M2 body weight of a cytostatic as characterized above.
The combinations of a selenium compound and a cytostatic or several cytostatics can be administered in solid or liquid form. The application may be oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous), or by inhalation. They may be administered together with conventional adjuvants, carriers and/or diluents.
The solid forms of application comprise tablets, capsules, powders, pills, pastilles, suppositories and granular forms of administration. They may also include additives, such as flavors, dyes, diluents, softeners, binders, preservatives, blasting agents and/or enclosing materials.
Liquid forms of administration include solutions, suspensions and emulsions. These may also be offered together with the above-mentioned additives.